This invention relates to compositions and methods comprising xe2x80x9clymphotoxin-xcex2 receptor blocking agentsxe2x80x9d, which block lymphotoxin-xcex2 receptor signalling. Lymphotoxin-xcex2 receptor blocking agents are useful for treating lymphocyte-mediated immunological diseases, and more particularly, for inhibiting Th1 cell-mediated immune responses. This invention relates to soluble forms of the lymphotoxin-xcex2 receptor extracellular domain that act as lymphotoxin-xcex2 receptor blocking agents. This invention also relates to the use of antibodies directed against either the lymphotoxin-xcex2 receptor or its ligand, surface lymphotoxin, that act as lymphotoxin-xcex2 receptor blocking agents. A novel screening method for selecting soluble receptors, antibodies and other agents that block LT-xcex2 receptor signalling is provided.
The pattern of cytokines released at the onset of an immune challenge can affect the subsequent choice of which immune effector pathways are activated. The choice between immune effector mechanisms is mediated by CD4-positive helper T lymphocytes (T helper cells or Th cells). Th cells interact with antigen-presenting cells (APCs), which display peptide fragments of processed foreign antigen in association with MHC class II molecules on their surfaces. Th cells are activated when they recognize particular epitopes of a foreign antigen displayed on the appropriate APC surface for which the Th cells express a specific receptor. Activated Th cells, in turn, secrete cytokines (lymphokines) which activate appropriate immune effector mechanisms.
Th cells can activate diverse effector mechanisms, including killer T cell activation B cell antibody production and macrophage activation. The choice between effector mechanisms is mediated largely by which cytokines are produced by the activated Th cells.
Th cells can be divided into three subgroups based on their cytokine secretion patterns (Fitch et al., Ann. Rev. Immunol., 11, pp. 29-48 (1993)). These subgroups are called Th0, Th1 and Th2. In the mouse, non-stimulated xe2x80x9cnaivexe2x80x9d T helper cells produce IL-2. Short term stimulation leads to Th0 precursor cells, which produce a wide range of cytokines including IFN-xcex3, IL-2, IL-4, IL-5 and IL-10. Chronically-stimulated Th0 cells can differentiate into either Th1 or Th2 cell types, whereupon the cytokine expression pattern changes.
Some cytokines are released by both Th1 and Th2 cells (e.g., IL-3, GM-CSF and TNF). Other cytokines are made exclusively by one or the other Th cell subgroup. The specialized effects of T helper cell subgroups were first recognized in mouse. A similar subdivision of T helper cells also exists in humans (Romagnani et al., Ann. Rev. Immunol., 12, pp. 227-57 (1994)).
Th1 cells produce LT-xcex1, IL-2 and IFN-xcex3. In humans, the Th1 pattern of cytokine secretion has been generally associated with cellular immunity and resistance to infection. The Th1 cytokines tend to activate macrophages and certain inflammatory responses such as Type IV xe2x80x9cdelayed typexe2x80x9d hypersensitivity (see below). Th1 cytokines play an important role in cellular rejection of tissue grafts and organ transplants.
Th2 cells produce the cytokines IL-4, IL-5, IL-6 and IL-10. Th2 cytokines increase eosinophil and mast cell production and promote the full expansion and maturation of B cells (Howard et al., xe2x80x9cT cell-derived cytokines and their receptorsxe2x80x9d, Fundamental Immunology, 3d ed., Raven Press, New York (1993)). Th2 cytokines also enhance antibody production, including IgE antibodies associated with allergic responses and anti-graft antibodies. Th2 cells may also participate in immune suppression and tolerance to persistent antigens.
Th1- and Th2-associated cytokines play a role in certain hypersensitivity responsesxe2x80x94inappropriate or disproportionate immune responses evoked upon contact with a previously encountered antigen. There are four recognized types of hypersensitivity (Roitt et al., Immunology, pp. 19.1-22.12 (Mosby-Year Book Europe Ltd., 3d ed. 1993)).
Type I xe2x80x9cimmediate hypersensitivityxe2x80x9d involves allergen-induced Th2 cell activation and Th2 cytokine release. The Th2 cytokine IL-4 stimulates B cells to undergo isotype switching to produce IgE, which activates mast cells to produce acute inflammatory reactions such as those which lead to eczema, asthma and rhinitis.
Types II and III hypersensitivity are caused by IgG and IgM antibodies directed against cell surface or specific tissue antigens (Type II) or soluble serum antigens (Type III). These types of hypersensitivity reactions are not thought to be mediated by Th cells.
Type IV xe2x80x9cdelayed typexe2x80x9d hypersensitivity (DTH) is Th1 cell mediated. DTH reactions take more than 12 hours to develop and are referred to as xe2x80x9ccell-mediatedxe2x80x9d because they can be transferred between mice by transferring Th1 cells but not serum alone. Type IV DTH responses are generally classified into three types: contact, tuberculin-type and granulomatous hypersensitivity.
Many cell-mediated responses that can cause disease are inducible in healthy mice by transferring lymphocytes from a diseased mouse (e.g., insulin-dependent diabetes and experimental autoimmune encephalitis). This feature distinguishes Type IV DTH from the other three types of hypersensitivity, which are humoral immune responses caused primarily by antibodies which can be transferred in cell-free serum.
T helper cells also participate in the regulation of de novo immunoglobulin isotype switching. Different Th subsets may influence the relative proportion of immunoglobulins of a given isotype produced in response to immune challenge. For example, the Th2 cytokine IL-4 can switch activated B cells to the IgG1 isotype and suppress other isotypes. As discussed above, IL-4 also activates IgE overproduction in type I hypersensitivity reactions. The Th2 cytokine IL-5 induces the IgA isotype. These Th2 cytokine effects on isotype switching are counter-balanced by IFN-xcex3 produced by Th1 cells.
The differential patterns of cytokines secreted by Th1 and Th2 cells appear to direct a response towards different immune effector mechanisms. The switch that activates either a cell-mediated or humoral effector mechanism is sensitized by cross-suppression between Th1 and Th2 cells: IFN-xcex3 produced by Th1 cells inhibits Th2 cell proliferation and Th2 cell-secreted IL-10 appears to reduce cytokine secretion from Th1 cells.
Depending on the relative affinities of the cytokines for their molecular targets, the Th1 and Th2 negative regulatory circuits may amplify the effects of small concentration differences between Th1 and Th2 cytokines. An amplified Th1 or Th2 cytokine signal may trigger the switch between cell-mediated or humoral effector mechanisms based on small changes in the relative concentrations of Th1 and Th2 cytokines. The ability to control this switch by modulating the relative concentrations of Th1 and Th2 cytokines would be useful for treating imbalances in a variety of Th1 and Th2 cell-dependent immune responses which can lead to immune disorders and diseases.
Pathological Th1 responses are associated with a number of organ-specific and systemic autoimmune conditions, chronic inflammatory diseases, and delayed type hypersensitivity reactions. As discussed above, Th1 responses also contribute to cellular responses leading to grafted tissue and transplanted organ rejection.
The treatment of these various Th1 cell-based immunological conditions to date has generally employed immunomodulatory and immuno suppressive agents as well as a number of drugs with poorly characterized mechanisms (e.g., gold or penicillamine). Three general immunosuppressive agents used currently are steroids, cyclosporine and azathioprine.
Steroids are pleiotropic anti-inflammatory agents which suppress activated macrophages and inhibit the activity of antigen presenting cells in ways which reverse many of the effects of the Th1 cytokine IFN-xcex3. Cyclosporinexe2x80x94a potent immunosuppressive agentxe2x80x94suppresses cytokine production and reduces the expression of IL-2 rerectors on lymphocytes during their activation. Azathioprine is an anti-proliferative agent which inhibits DNA synthesis. These non-specific immunosuppressive agents are generally required in high doses which increase their toxicity (e.g. nephro- and hepatotoxicity) and cause adverse side effects. They are thus unsuitable for long term therapies.
To address the problems caused by conventional treatments with non-specific immunosuppressive agents, many current therapeutic strategies aim at suppressing or activating selective aspects of the immune system. An especially attractive goal is the manipulation of the balance between Th1 and Th2 cytokines to shift the balance between cell-mediated and humoral effector mechanisms.
To accomplish a shift between cell-mediated and humoral effector mechanisms, it would be useful to be able to modulate the activity of a molecule that can shift the relative activities of Th1 and Th2 cell subclasses. Candidates for such molecules include the cytokines and their receptors. Recent data suggest that LT-xcex1, IL-12, IFN-xcex1 and IFN-xcex3 favor the development of Th1 responses, whereas IL-1 and IL-4 polarize a response towards a Th2 effector mechanism (Romagnani et al., Ann. Rev. Immunol., 12 , pp. 227-57 (1994)).
Many of the Th cell cytokines are pleiotropic regulators of immune development and function, and inhibiting their production would have deleterious effects on non-T cell mediated responses. A desirable and effective target for selectively modulating the choice between Th1 and Th2 effector mechanisms has not been identified.
The present invention solves the problems referred to above by providing pharmaceutical compositions and methods for treating immunological diseases by inhibiting lymphotoxin-xcex2 receptor (LT-xcex2-R) signalling using lymphotoxin-xcex2 receptor blocking agents. More particularly, the compositions and methods comprising LT-xcex2-R blocking agents are useful for inhibiting Th1 cell-mediated immune responses such as, for example, inflammatory bowel syndrome.
In one embodiment, soluble forms of the lymphotoxin-xcex2 receptor extracellular domain that act as LT-xcex2-R blocking agents are provided. The preferred compositions and methods of this embodiment comprise a recombinant lymphotoxin-xcex2 receptor fusion protein that has the LT-xcex2-R extracellular ligand binding domain fused to an immunoglobulin constant heavy chain domain. More preferably, the LT-xcex2-R ligand binding domain is fused to a human IgG Fc domain.
In another embodiment of this invention, antibodies that act as LT-xcex2-R blocking agents are provided. Preferred compositions and methods of this embodiment comprise one or more antibodies directed against the lymphotoxin-xcex2 receptor. More preferably, the antibody is a monoclonal antibody. Other preferred compositions and methods of this embodiment comprise one or more antibodies directed against surface lymphotoxin. More preferably, the antibody is a monoclonal antibody directed against lymphotoxin-xcex2.
This invention further provides a novel screening process for selecting LT-xcex2-R blocking agentsxe2x80x94such as soluble forms of the LT-xcex2-R, anti-LT Abs and anti-LT-xcex2-R Abs. This screening process involves performing tumor cell cytotoxicity assays that monitor LT-xcex2-R signalling. The assay makes use of the increased sensitivity of human adenocarcinoma cells to ligand- or antibody-induced LT-xcex2-R signalling in the presence of an LT-xcex2-R activating agent (such as LT-xcex11/xcex22) in a tumor cytotoxicity assay.
LT-xcex2-R blocking agents inhibit the cytotoxic effects of LT-xcex1/xcex2heteromeric complexes (or other LT-xcex2-R activating agents) on tumor cells. The procedure used to test putative LT-xcex2-R blocking agents is exemplified for the case of anti-LT-xcex2-R antibodies (in the presence of the LT-xcex2-R activating agents LT-xcex11/xcex22) and comprises the following steps:
1) Tumor cells (e.g., HT29 human adenocarcinoma cells) are cultured for several days in media containing IFN-xcex3 and purified LT-xcex11/xcex22 in the presence or absence of the particular anti-LT-xcex2-R Ab being assayed;
2) The cells are treated with a dye that stains living cells; and
3) The number of stained cells is quantitated to determine the fraction of tumor cells killed in the presence of LT-xcex11/xcex22, IFN-xcex3 and the test anti-LT-xcex2-R Ab in each sample. Alternatively, the number of surviving cells can be determined by any of a number of well-known assays which measure cell viability, such as 3H-thymidine incorporation into DNA. An anti-LT-xcex2-R Ab (or an Ab combination) that decreases the percentage of tumor cells killed in this assay by at least 20% is a LT-xcex2-R blocking agent within the scope of this invention.
This cytolytic assay may be performed using LT-xcex1/xcex2 heteromeric complexes and other LT-xcex2-R activating agents, either alone or in combination. The assay can also be adapted as required to identify new LT-xcex2-R blocking agents.